Knowledge Base

A standard H&E staining protocol is provided below.  It applies to either a manual or automated staining procedure.  While it may not be ideal for your laboratory, it can be used as a starting point.  The final color rendition of your H&E stain should be determined by working with your pathologists.  This will consequently make their job easier.  Each day, once the H&E stain set up is completed, you should run down one test slide to confirm that the staining is optimal.  This also will help you to document quality control procedures.

What exactly is a routine “H&E”?  And what makes it routine?  The first question is easy.  “H” stands for ”hematoxylin” and “E” stands for “eosin”.  Both are dyes used to stain tissue sections in histology.  However, the procedure for correctly applying this combination of stains to tissue sections is far from routine.

In the histology world, the mere mention of a “silver stain” may be the cause of panic and uncertainty with regard to the performance of the stain, and the quality of the final resulting microscope slide.  All other special stains, with few exceptions, are relatively easy and straightforward to perform; not so with silver stains.

The staining of microorganisms in histology can be challenging. Filamentous fungi and associated conidia are more easily demonstrated as they are visible under light microscopy when stained with periodic acid Schiff’s (PAS).

Fungi include molds, yeasts and higher fungi. All fungi are eukaryotic and have sterols but not peptidoglycan in their cell membrane. Their cell walls are composed of cellulose; the same building blocks that plants use.

There are some pathological conditions that exist that cannot be accurately diagnosed by examining hematoxylin and eosin (H&E) stained slides alone. In such cases, the pathologist may order immunohistochemical (IHC) stains to help render a diagnosis.

When staining sections for the presence of carbohydrates, the two main classes under investigation are glycogen and mucins.  Mucins include substances referred to as mucopolysaccharides, mucosubstances, glycoproteins and glycoconjugates.

1. incomplete fixation
2. improper embedding
3. incomplete dehydration
4. incomplete infiltration with paraffin